Critical Care

27 Pages·2008·0.38 MB·English

Critical Care
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Use of the probiotic Lactobacillus plantarum 299 to reduce pathogenic bacteria
in the oropharynx of intubated patients: a randomised controlled open pilot
study
Critical Care 2008, 12:R136 doi:10.1186/cc7109
Bengt Klarin ([email protected])
Goran Molin ([email protected])
Bengt Jeppsson ([email protected])
Anders Larsson ([email protected])
ISSN 1364-8535
Article type Research
Submission date 25 June 2008
Acceptance date 6 November 2008
Publication date 6 November 2008
Article URL http://ccforum.com/content/12/6/R136
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Use of the probiotic Lactobacillus plantarum 299 to reduce pathogenic
bacteria in the oropharynx of intubated patients: a randomised
controlled open pilot study

Bengt Klarin1, Göran Molin2, Bengt Jeppsson3, Anders Larsson4

1Department of Anaesthesiology and Intensive Care, University Hospital, SE-221 85 Lund,
Sweden
2Applied Nutrition and Food Chemistry, Lund University, Box 117, SE-221 00 Lund, Sweden
3Department of Surgery, University Hospital, SE-205 02 Malmö, Sweden
4Department of Anaesthesiology and Intensive Care, Aalborg Hospital, Århus University
Hospitals, DK-9000 Aalborg, Denmark

Corresponding author: Bengt Klarin, [email protected]

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Abstract
Introduction: Ventilator-associated pneumonia (VAP) is usually caused by aspiration of
pathogenic bacteria from the oropharynx. Oral decontamination with antiseptics, such as
chlorhexidine (CHX) or antibiotics, has been used as prophylaxis against this complication.
We hypothesised that the probiotic bacteria Lactobacillus plantarum 299 (Lp299) would be as
efficient as CHX in reducing the pathogenic bacterial load in the oropharynx of tracheally
intubated, mechanically ventilated, critically ill patients.
Method: Fifty critically ill patients on mechanical ventilation were randomised to either oral
mechanical cleansing followed by washing with 0.1% CHX solution or to the same cleansing
procedure followed by oral application of an emulsion of Lp299. Samples for microbiological
analyses were taken from the oropharynx and trachea at inclusion and at defined intervals
thereafter.
Results: Potentially pathogenic bacteria that were not present at inclusion were identified in
oropharyngeal samples from eight of the patients treated with Lp299 and 13 of those treated
with CHX (p = 0.13). Analysis of tracheal samples yielded similar results. Lp299 was
recovered from the oropharynx of all patients in the Lp299 group.
Conclusion: In this pilot study, we found no difference between the effect of Lp299 and CHX
used in oral care procedures, when we examined the effects of those agents on colonisation of
potentially pathogenic bacteria in the oropharynx of intubated, mechanically ventilated
patients.

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Introduction

Ventilator-associated pneumonia (VAP) is a common complication in intubated, mechanically
ventilated patients in intensive care units (ICUs). VAP is connected to longer ICU and
hospital stays, additional costs and high mortality, and the risk of developing this condition
increases by 1% with each additional day of mechanical ventilation [1,2].

The major cause of VAP is aspiration of either microorganisms from the oropharynx or
fragments of biofilms from the endotracheal tube. Formation of such biofilms can be delayed,
but not prevented, by the use of tubes with special coatings [3]. Selective decontamination
using antibiotics in the oral cavity alone [4-6] or throughout the gastrointestinal (GI) tract
[7,8], has been shown to lower the incidence of VAP and reduce mortality. However, the use
of such procedures is limited due to the risk of bacteria developing resistance to the antibiotics
[9-10]. In recent meta-analyses, it was concluded that oral decontamination with
chlorhexidine (CHX) could prevent VAP [11], but that strategy does not reduce the time on
the ventilator, the length of stay (LOS) in the ICU or rates of mortality [12]. Thus, there is a
need for alternative approaches to lower the oropharyngeal load of pathogenic
microorganisms as a means of decreasing the risk of VAP.

For decades, probiotics have been given enterally to improve the microbiotic flora in the GI
tract. However, in recent years orally administered probiotics have also been shown to reduce
the number of bacteria and yeast in biofilms on vocal prostheses [13,14]. Therefore, we
hypothesised that swabbing the oral mucosa with probiotics would be an effective (and
microbiologically attractive) method of reducing pathogenic oral microorganisms in
tracheally intubated, mechanically ventilated, critically ill patients.
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The primary aim of the present pilot study was to evaluate the feasibility and safety of an oral
care procedure using the probiotic Lactobacillus plantarum 299 (Lp299) (DSM 6595) in this
patient category.Like the genomically closely related strain L plantarum 299v (DSM 9843),
Lp299 has been shown to adhere to the mucosa throughout the GI tract [15-17]. Another
objective of this preliminary investigation was to obtain an estimate of the number of patients
needed for a definitive study examining the effectiveness of oral Lp299 in reducing the
incidence of VAP.

Materials and methods

The study was approved by the Human Ethics Committee of Lund University and was
performed in compliance with the Helsinki Declaration. Good clinical practice and the
International Conference on Harmonisation Guidance were applied and the investigation was
carried out in the ICU of the Department of Anaesthesiology and Intensive Care, University
Hospital, Lund, Sweden. Informed consent was obtained from the patients or their next of kin.
Further consent was not obtained from patients as they had recovered, as this was not required
by the Human Ethics Committee.

The patients were randomised into groups of 10 to receive either the department’s standard
oral treatment (the control group) or the study treatment with Lp299 (the Lp group). The day
of inclusion was designated day 1. To be included in the study, patients had to fulfil the
following criteria: 18 years of age or older; critically ill with an anticipated need for
mechanical ventilation of at least 24 hours; not moribund; not suffering from pneumonia at
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admission; no fractures in the facial skeleton or the base of the skull; no oral ulcers; not
immune deficient; not a carrier of HIV or viral hepatitis.

After screening, patients were included when ventilation and circulation had been stabilised
and before the first oral care procedure. Oral care was performed twice a day. The control
group was treated according to the department’s standard protocol: dental prostheses were
removed; secretions were removed by suction; teeth were brushed using toothpaste (Zendium,
Opus Health Care, Malmö, Sweden); all mucosal surfaces were cleansed with swabs that had
been moistened with a 1 mg/ml CHX solution (Hexident, Ipex, Solna, Sweden). In the Lp
group the initial mechanical steps were the same as in the control group, but the subsequent
cleansing was instead performed with gauze swabs soaked in carbonated bottled water, after
which Lp299 was applied to the mucosal surface of the oral cavity. This was performed using
two gauze swabs (one for each side of the oral cavity), which had been allowed to absorb 10
ml of a solution containing a total of 1010 colony-forming units (CFU) of Lp299. Excess
suspension was not removed. In both groups, when necessary between the oral care
procedures, secretions were removed by suctioning and gauze swabs moistened with
carbonated bottled water were used to wipe off remaining secretions.

Cultures were taken from the oropharynx and from the trachea at inclusion. Sampling was
repeated before the oral care procedures on days 2, 3, 5, 7, 10, 14 and 21 in patients that were
still mechanically ventilated. If a patient was extubated on a non-culture day, cultures were
taken before the extubation. One set of cultures was analysed according to normal routines at
the Department of Clinical Microbiology, University Hospital. Another set was sent blinded
to the research laboratory at Probi AB, Lund, Sweden for identification and quantification of
total CFU of lactobacilli and identification of Lp299. Viable counts of all lactobacilli were
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obtained on Rogosa agar (Oxoid, Basingstoke, Hampshire, England) incubated anaerobically
at 37oC for three days. Colonies suspected to be Lp299 (large, creamy white-yellowish and
somewhat irregular in shape) were selected and identified by randomly amplified
polymorphic DNA typing [18].

The patients were placed in a semi-recumbent position and were ventilated in pressure control
or pressure support mode by a Servo ventilator (Maquet AB, Sweden) via a heat moisture
exchange filter (Barrierbac “S”, Mallinckrodt DAR, Mirandola, Italy). A closed suction
system (TRACH-Care 72, Ballard Medical Products, Draper, UT, USA) was used. The
patients inhaled 2.5 mg salbutamol (GlaxoSmithKline, Solna, Sweden) and 0.5 mg
ipratropium (Boehringer Ingelheim, Stockholm, Sweden) every six hours.

Chest radiographs were obtained after tracheal intubation and thereafter when clinically
indicated. Lung function was assessed using the Lung Injury Score (LIS) [19]. Blood gases
were obtained at least three times a day and were analysed at the ICU. Samples for white
blood cell (WBC) counts and C-reactive protein (CRP) were collected daily and analysed at
the hospital clinical chemistry laboratory.

Enteral nutrition was started and increased according to the department’s protocol. The
amount of enteral formula given and the total volume of other enterally administered fluids
were recorded. All patients received intravenous ezomprazol (Astra Zeneca, Södertälje,
Sweden) as stress ulcer prophylaxis from admission until enteral nutrition was fully
established (ie, for three to four days).

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The study was neither intended nor powered for assessment of differences in the frequency of
VAP. However, it was aimed at obtaining a basis for estimating the number of patients needed
for a larger investigation in which VAP also constitutes a parameter. The following criteria
were used to identify VAP: a new, persistent or progressive infiltrate on chest radiograph
combined with at least three of the other four criteria; a purulent tracheal aspirate; positive
culture of tracheal aspirates occurring after 48 hours of mechanical ventilation; rectal or urine
bladder temperature higher than 38.0oC or less than 35.5oC; WBC count more than 12 or less
than 3 [4,20].

Statistics
Because no previous investigation has examined the effect of probiotics in this context, we
estimated that 20 patients in each group would be sufficient to assess the safety, important
positive effects and possible side effects, and to give an indication of the number of patients
that would be needed in a definitive study. Statistical methods were chosen after consulting a
biostatistician, and the statistical analyses were performed using Statistica 6.0 (StatSoft,
Tulsa, OK, USA). Student’s t-test was used for the daily comparisons (days1 to 9) of the
parameters. Fisher’s exact test was employed to compare the results of microbiological
cultures. p< 0.05 was considered significant.

Results

After screening, 50 patients were included in the study. Consent was withdrawn by two
patients and another three were transferred to other ICUs shortly after inclusion. For one
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patient in the control group, samples were obtained only at inclusion. Altogether, 23 patients
in the Lp group and 21 in the control group completed the study.

All patients were orotracheally intubated. Two in each group were reintubated, and two in the
Lp group and one in the control group were tracheotomised (on days 3, 16 and 3,
respectively). The proportion of patients receiving enteric nutrition and the volumes given
were similar in the two groups. The patients in both groups were treated with antibiotics at the
discretion of the attending physician and changes were made in compliance with culture
results. Cefuroxime was the most common antibiotic used in both groups, followed by
imipenem. Three patients in each group received piperacillin/tazobactam, and other
antibiotics or combinations were administered to some patients in each of the two groups.
Three patients did not receive any antibiotics at admission, and one of those three was not
treated with antibiotics during the stay in the ICU. Ten patients in each group received
corticosteroids for one or more days.

As indicated in Table 1, there were no significant differences in age or gender between the
groups. Also, the admission diagnoses were similar in the two groups, as were the Acute
Pathophysiology and Chronic Health Evaluation (APACHE) II scores. Some differences were
found in the Sequential Organ Failure Assessment (SOFA) scores in favour of the Lp patients
(data not shown). The two groups did not differ significantly with regard to the number of
ventilator days, LOS, or ICU or in-hospital mortality (Table 1). No deaths were caused by
respiratory complications and no additional deaths occurred within six months.

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No differences in WBC counts were found between the groups. Furthermore, the groups did
not differ with regard to changes in CRP, although the absolute values were higher for the
controls on day 3.

No significant differences between the two groups were found when considering
microbiological findings of the oropharyngeal and tracheal samples taken at inclusion. The
same species were identified in samples from both the oropharynx and the trachea of six Lp
patients and three controls. Subsequent oropharyngeal samples from eight Lp patients and
from 13 controls contained enteric species that had not been present in the inclusion samples
from those patients (p = 0.13) (Table 2). Two or three emerging species (enterococci species
and enterobacteriaceae species) were found in two patients in the Lp group and seven control
patients (Figure 1). Culture analysis of the tracheal samples identified emerging species in
seven Lp patients and nine controls. Other comparisons of the culture results were similar.
Figure 2 shows the distribution of the positive cultures according to study day and sampling
site.

Lp299 was found in the oropharyngeal samples from all of the patients in the Lp group (21 of
23 patients on day 2). In addition, Lp299 was identified in the tracheal secretion samples from
13 of the patients in the Lp group (56%), and enteric bacteria were also found in six of those
subjects. Five patients in the Lp group died in the ICU, and Lp299 was identified in the
tracheal samples from one of those individuals, whereas no enteric bacteria were recovered
from the trachea of any of those five patients.

Considering patients in both groups, a comparison of those with positive findings and those
with negative findings in cultures of tracheal secretions (results reported by the microbiology
9